博碩士論文 111827014 詳細資訊



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姓名 簡逢原(Feng-Yuan Chien)  查詢紙本館藏   畢業系所 生物醫學工程研究所
論文名稱 使用3D列印技術建構仿生耳蝸微器官 應用於聽損相關研究
(Using 3D Printing Technology to Construct Biomimetic Cochlear Organoids for Hearing Loss-related Research)
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摘要(中) 根據世界衛生組織(World Health Organization, WHO)在 2022 年 3 月所發布的聽力統計表明，現今全球有近5億的人口患有聽力受損(Hearing Loss)這項疾病，而在2050年時患病人口數將會成長到10億左右，原因在於各國科技的快速發展及醫療水平逐漸提升，導致全球人口的平均壽命延長並且步入高齡化社會(Aging Society)，同時針對於人類的感覺器官來說，聽覺的衰退是最為快速，若情況嚴重甚至會影響與人進行社交的能力，因此需要即刻提出有效的治療方案，依聽損症狀主要可以分為三大類，分別是:傳導型聽損(Conductive Hearing Loss)、感音神經型聽損Sensorineural Hearing Loss)及混和型聽損(Mixed Hearing Loss)，其中又以感音神經型聽損為最大宗，約佔有8成左右的比例，造成該聽損的可能原因是長期處在高分貝的噪音環境、服用具有耳毒性的藥物、遺傳、老化、病毒等種種因素，進而破壞位處內耳深處的聽毛細胞(Inner Ear Hair Cell)，通常對哺乳動物(Mammal)的聽毛細胞造成的損傷是屬於不可逆的，目前所使用的治療方案為助聽器(Hearing Aids)及人工電子耳(Cochlear Implants)等醫療輔具來維持基本的聽力行為，而在藥物治療的部分目前仍在進行臨床試驗還無法實際應用於聽損治療。在本研究中，我們將探討三個主要方面，分別為細胞在動靜態環境下進行三維耳蝸細胞成熟化的細胞培養、仿生耳蝸的影像重構及3D列印，以及最終的新型體外耳蝸
模型。 在第一部分，我們以細胞培養為實驗的主軸。本研究以P0~P2的初生鼠為主要研究對象，許多文獻指出新生哺乳動物的耳蝸細胞具有再生能力，通常這種能力會隨著出生時間的增加而急劇下降，在出生後的三天至一周內即消失，因此我們希望透過這樣的方式，提取具有再生能力的耳蝸細胞，並且結合生物3D列印技術，將這些細胞混入生物墨水中進行列印，提供三維培養環境之外添加適當的培養基EFICVP6來促進細胞增生，以及分化試劑LYC進行耳蝸細胞的成熟化，以成功在體外重新分化出新生的聽毛細胞(Hair Cell)。第二部分將以耳蝸影像重構為主要研究核心，耳蝸內部結構相對複雜，目前尚無比較完整的體外耳蝸模型，且臨床實驗多半以豬隻或老鼠為練習對象，但在個體及耳蝸顯微結構上仍存在顯著差異，同時在手術中醫師難以確認電子耳實際穿入的位置及是否對耳蝸內部造成損傷，影響內耳治療的準確性和安全性。因此，本研究希望透過影像重建結合3D列印，能夠完整復刻出新型體外耳蝸模型，以提高手術準確性和安全性。第三部分將會整合前兩部分，進行耳蝸及電子耳的影像重構。主要目的是提供耳科醫師更完整的手術練習模型，同時在電子耳上搭載治療內耳的藥物，隨後，將搭載藥物的電子耳進行穿入的動作，模擬手術實際穿入過程，這樣的配套措施不但成為內耳治療藥物篩選模型，也能為未來聽損治療相關研究提供更完整的研究方案。
摘要(英) According to the World Health Organization (WHO) statistics released in March 2022,nearly 500 million people worldwide suffer from hearing loss. By 2050, this number is expected to grow to around 1 billion due to the rapid development of technology and the gradual improvement of medical standards, which have led to an increase in global life expectancy and an aging society. Hearing decline is the most rapid among human sensory organs, and severe cases can affect the ability to socialize, necessitating the immediate proposal of effective treatment plans. Hearing loss symptoms can be divided into three main categories: conductive hearing loss, sensorineural hearing loss, and mixed hearing loss.
Sensorineural hearing loss is the most prevalent, accounting for about 80% of cases. Possible causes include long-term exposure to high-decibel noise, use of ototoxic drugs, genetics, aging, and viruses, which can damage the inner ear hair cells. Damage to these cells in mammals is usually irreversible. Current treatments include the use of hearing aids and cochlear implants to maintain basic hearing functions, while pharmaceutical treatments are still undergoing clinical trials and cannot yet be practically applied to hearing loss treatment. This study will explore three main aspects: three-dimensional cochlear cell maturation in dynamic and static environments, image reconstruction and 3D printing of a bionic cochlea,
and the development of a novel in vitro cochlear model.
In the first part, the focus is on cell culture experiments. This research uses newborn mice (P0-P2) as the main subjects. Literature indicates that cochlear cells of newborn mammals possess regenerative abilities, which typically diminish rapidly with age, disappearing within three days to a week after birth. Therefore, we aim to extract cochlear cells with regenerative capabilities and use bio-3D printing technology to mix these cells into
bio-ink for printing, providing a three-dimensional culture environment. We will add appropriate culture media, EFICVP6, to promote cell proliferation, and differentiation reagent
LYC for cochlear cell maturation, successfully differentiating new hair cells in vitro. The second part focuses on cochlear image reconstruction. The internal structure of the cochlea is relatively complex, and currently, there is no complete in vitro cochlear model.
Clinical experiments often use pigs or mice as practice subjects, but significant differences exist in individual and cochlear microstructures. During surgery, it is challenging for physicians to confirm the actual position of the cochlear implant and whether it causes internal cochlear damage, affecting the accuracy and safety of inner ear treatments. Therefore, this study aims to create a new in vitro cochlear model through image reconstruction
combined with 3D printing, improving surgical accuracy and safety. The third part will integrate the first two parts, focusing on cochlear and cochlear implant image reconstruction. The main goal is to provide otologists with a more complete surgical practice model. Additionally, we aim to load therapeutic drugs for inner ear treatment
onto the cochlear implant. Subsequently, the drug-loaded cochlear implant will be inserted to simulate the surgical insertion process. This approach serves as a drug screening model for inner ear treatment and provides a more comprehensive research plan for future hearing loss
treatment research.
關鍵字(中) ★ 聽力損失
★ 三維培養
★ 生物支架
★ 3D列印
★ 聽毛細胞再生
★ 耳蝸重構		 關鍵字(英) ★ Hearing Loss
★ Three-dimensional Culture
★ Bio-scaffold
★ 3D-Printing
★ Hair Cells Regeneration
★ Cochlear Reconstruction
論文目次 目錄
一. 研究背景暨文獻回顧 1
1-1 聽力損失簡介 1
1-1-1 疾病數據統計 1
1-1-2 聽力損失的定義 1
1-1-3 聽力損失的原因 2
1-1-4 聽力損失的類型 2
1-1-5 現行的治療方式 5
1-2 耳朵的解剖結構及生理功能 7
1-2-1 耳朵的構造及聲音傳導路徑 7
1-2-2 耳蝸 (Cochlea) 9
1-2-3 外淋巴液(Perilymph)和內淋巴液 (Endolymph) 10
1-2-4 柯蒂氏器 (Organ of Corti) 10
1-2-5 聽毛細胞 (Hair Cells) 11
1-2-6 支持細胞 (Supporting Cells) 12
1-3 不同物種間的耳蝸構型差異 14
1-3-1 鳥類 (Birds) 14
1-3-2 兩棲動物類 (Amphibians) 14
1-3-3 哺乳類 (Mammals) 15
1-4 細胞培養技術 16
1-4-1 二維細胞培養 (2D Cell Culture) 16
1-4-2 三維細胞培養 (3D Cell Culture) 17
1-4-3 靜態細胞培養(Static Cell Culture) 20
1-4-4 動態細胞培養(Dynamic Cell Culture) 21
1-5 3D列印技術 23
1-5-1 立體光刻3D列印 24
1-5-2 金屬3D列印 25
1-5-3 生物3D列印 27
二. 研究動機與實驗目的 30
三.材料與實驗方法 31
3-1 使用儀器與耗材 31
3-1-1 藥品清單 31
3-1-2 耗材清單 32
3-1-3 儀器清單 33
3-1-4 培養液成分表 33
3-2 採集耳蝸前驅細胞(Cochlear Progenitor Cells, CPCs) 34
3-3 生物墨水的製備(Prepare the Bio-ink) 35
3-3-1 膠體的配置 35
3-3-2 製作生物墨水 36
3-3-3 3D列印參數設定 37
3-4 細胞培養 40
3-4-1 二維細胞培養-Pre-plate 40
3-4-2 三維細胞培養-靜態 41
3-4-3 三維細胞培養-動態 42
3-5 定性分析(Qualitative Test) 44
3-5-1 共軛焦螢光顯微鏡(Confocal Microscopy) 44
3-5-2 細胞染色(Staining) 45
3-5-3 毛細胞特異性標記物染色(Hiar Cell Specific Marker) 47
3-6 定量分析(Quantitative Test) 48
3-7 影像重構(Image Reconstruction) 48
3-7-1 豬耳蝸(Porcine Cochlea)影像重構 48
3-7-2 人工電子耳(Cochlear Implant)影像重構 49
四. 結果與討論 51
4-1 二維細胞培養-Pre-plate 51
4-1-1 PP1，DMEM-HG & LG 51
4-1-3 PP3-HG & LG 53
4-1-4 PP4-EFICVP6 & LYC 54
4-2 三維細胞培養-3D-Printing(Static) 56
4-2-1 3D-Printing(Static)-EFICVP6(14-DIV) 56
4-2-2 3D-Printing(Static)-LYC(14-DIV) 57
4-3 三維細胞培養-3D-Printing(Dynamic) 60
4-3-1 支架及圓盤的設計 60
4-3-2 動態細胞培養實驗設計時間軸 61
4-4 Q-PCR 62
4-4-1 Q-PCR Protocal 62
4-4-2 Q-PCR的溶液配置及測定參數 63
4-4-3 Q-PCR的結果與分析 64
4-5 Staining & Confocal 66
4-5-1 2D Staining(Pre-plate) 66
4-5-2 3D Cell Culture Staining (Static) 69
4-5-3 3D Cell Culture Staining (Dynamic) 72
4-5-4 比較不同培養條件細胞的分化程度 73
4-5-5 3D Cell Culture Staining( On Collagen Surface) 74
4-5-6 Compared The Differences of Cell Nuclei 76
4-5-7 神經樣貌(Neural-like)的細胞 77
4-5-8 3D Cell Culture in Matrigel®(Ref.) 78
4-5-9 比較不同培養系統之間的差異 79
4-6 Image Reconstructed 80
4-6-1 Porcine Cochlear Image Reconstructed 80
4-6-2 Cochlear Implant Image Reconstruction 82
五. 結論 84
六. 參考文獻 85
參考文獻 1. Organization, W.H. Deafness and hearing loss. 2021; Available from: https://wd.vghtpe.gov.tw/ent/Fpage.action?fid=4304.
2. Aging, N.I.o. Hearing Loss: A Common Problem for Older Adults. Available from: https://www.nia.nih.gov/health/hearing-and-hearing-loss/hearing-loss-common-problem-older-adults.
3. Sooriyamoorthy, T. and O. De Jesus, Conductive Hearing Loss, in StatPearls. 2024, StatPearls Publishing
Copyright © 2024, StatPearls Publishing LLC.: Treasure Island (FL).
4. Clason, H.H.-D. Causes of sensorineural hearing loss (SNHL) Aging, genetics and noise exposure all play a role. January 23, 2023; Available from: https://www.healthyhearing.com/report/50276-Common-causes-of-sensorineural-hearing-loss.
5. Tanna, R.J., J.W. Lin, and O. De Jesus, Sensorineural Hearing Loss, in StatPearls. 2024, StatPearls Publishing
Copyright © 2024, StatPearls Publishing LLC.: Treasure Island (FL).
6. Fieux, M., F. Podeur, and S. Tringali, Mixed hearing loss with normal eardrum. Eur Ann Otorhinolaryngol Head Neck Dis, 2021. 138(4): p. 299-300.
7. Young, A. and M. Ng, Otoacoustic Emissions, in StatPearls. 2024, StatPearls Publishing
Copyright © 2024, StatPearls Publishing LLC.: Treasure Island (FL).
8. Wahid, N.W.B., C.J. Hogan, and M. Attia, Weber Test, in StatPearls. 2024, StatPearls Publishing
Copyright © 2024, StatPearls Publishing LLC.: Treasure Island (FL).
9. Szyfter, W., et al., Conductive hearing loss after surgical treatment of otosclerosis - long-term observations. Otolaryngol Pol, 2020. 75(1): p. 1-6.
10. Otitis media with effusion. Pediatrics, 2004. 113(5): p. 1412-29.
11. 台北榮民總醫院耳鼻喉頭頸醫學部. 久治不癒的小兒中耳積水，該怎麼辦. 2023.
12. 藥師, 中.第.卷.第.羅. 突發性耳聾(SSNHL)之治療. 2012; Available from: https://www.cmuh.cmu.edu.tw/FileUploads/FileUpload/10112.
13. Davis, A., et al., Aging and Hearing Health: The Life-course Approach. Gerontologist, 2016. 56 Suppl 2(Suppl 2): p. S256-67.
14. Lieu, J.E.C., et al., Hearing Loss in Children: A Review. Jama, 2020. 324(21): p. 2195-2205.
15. Fuchs, J.C. and A.S. Tucker, Development and Integration of the Ear. Curr Top Dev Biol, 2015. 115: p. 213-32.
16. Hosoi, H., et al., Cartilage conduction as the third pathway for sound transmission. Auris Nasus Larynx, 2019. 46(2): p. 151-159.
17. edition., N.L.o.M.-N.n. The Semicircular Canals. Available from: https://www.ncbi.nlm.nih.gov/books/NBK10863/.
18. Thulasiram, M.R., J.M. Ogier, and A. Dabdoub, Hearing Function, Degeneration, and Disease: Spotlight on the Stria Vascularis. Front Cell Dev Biol, 2022. 10: p. 841708.
19. Salt, A.N. and K. Hirose, Communication pathways to and from the inner ear and their contributions to drug delivery. Hear Res, 2018. 362: p. 25-37.
20. Li, Y., et al., Endolymphatic Potential Measured From Developing and Adult Mouse Inner Ear. Front Cell Neurosci, 2020. 14: p. 584928.
21. Wan, G., G. Corfas, and J.S. Stone, Inner ear supporting cells: rethinking the silent majority. Semin Cell Dev Biol, 2013. 24(5): p. 448-59.
22. Kelley, M.W., Cochlear Development; New Tools and Approaches. Front Cell Dev Biol, 2022. 10: p. 884240.
23. Zhang, S., et al., Characterization of Strip1 Expression in Mouse Cochlear Hair Cells. Front Genet, 2021. 12: p. 625867.
24. Rask-Andersen, H., et al., Human cochlea: anatomical characteristics and their relevance for cochlear implantation. Anat Rec (Hoboken), 2012. 295(11): p. 1791-811.
25. Rubel, E.W., S.A. Furrer, and J.S. Stone, A brief history of hair cell regeneration research and speculations on the future. Hear Res, 2013. 297: p. 42-51.
26. McGovern, M.M., et al., Multiple supporting cell subtypes are capable of spontaneous hair cell regeneration in the neonatal mouse cochlea. Development, 2019. 146(4).
27. Jung, J.Y., et al., siRNA targeting Hes5 augments hair cell regeneration in aminoglycoside-damaged mouse utricle. Mol Ther, 2013. 21(4): p. 834-41.
28. Janesick, A., et al., Cell-type identity of the avian cochlea. Cell Reports, 2021. 34(12): p. 108900.
29. Schoffelen, R.L., J.M. Segenhout, and P. van Dijk, Mechanics of the exceptional anuran ear. J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 2008. 194(5): p. 417-28.
30. Fettiplace, R., Cochlear tonotopy from proteins to perception. BioEssays, 2023. 45(8): p. 2300058.
31. Robles, L. and M.A. Ruggero, Mechanics of the mammalian cochlea. Physiol Rev, 2001. 81(3): p. 1305-52.
32. Kapałczyńska, M., et al., 2D and 3D cell cultures - a comparison of different types of cancer cell cultures. Arch Med Sci, 2018. 14(4): p. 910-919.
33. Hess, M.W., et al., Chapter 27 - 3D Versus 2D Cell Culture: Implications for Electron Microscopy, in Methods in Cell Biology, T. Müller-Reichert, Editor. 2010, Academic Press. p. 649-670.
34. 臺大醫院-醫學研究部. 細胞的培養世界及微流體系統. Aug.10.2018; Available from: https://ntuhmc.ntuh.gov.tw/epaper-56th.htm.
35. Huang, L., et al. Biopolymer-Based Microcarriers for Three-Dimensional Cell Culture and Engineered Tissue Formation. International Journal of Molecular Sciences, 2020. 21, DOI: 10.3390/ijms21051895.
36. Lee, S.-Y., et al., In Vitro three-dimensional (3D) cell culture tools for spheroid and organoid models. SLAS Discovery, 2023.
37. 科技發展觀測平台-鄭采和、薛孝亭、洪立萍. 3D細胞培養發展趨勢與技術介紹. 2022/11/08; Available from: https://outlook.stpi.narl.org.tw/index/focus-news/4b114100840f0bc001845550d45d1155.
38. Hu, X., Z. Xia, and K. Cai, Recent advances in 3D hydrogel culture systems for mesenchymal stem cell-based therapy and cell behavior regulation. J Mater Chem B, 2022. 10(10): p. 1486-1507.
39. Biocoat, C. Micro Travel in Extracellular Matrix. Available from: https://www.unimed.com.tw/upload/200522_053950.
40. Huang, X., et al. Current Advances in 3D Dynamic Cell Culture Systems. Gels, 2022. 8, DOI: 10.3390/gels8120829.
41. Gao, W., et al., The Hollow Porous Sphere Cell Carrier for the Dynamic Three-Dimensional Cell Culture. Tissue Eng Part C Methods, 2022. 28(11): p. 610-622.
42. Hoyle, H.W., C.M.L. Stenger, and S.A. Przyborski, Design considerations of benchtop fluid flow bioreactors for bio-engineered tissue equivalents in vitro. Biomater Biosyst, 2022. 8: p. 100063.
43. Mehling, M. and S. Tay, Microfluidic cell culture. Curr Opin Biotechnol, 2014. 25: p. 95-102.
44. 國家衛生研究院. 晶片上的實驗室—微流體晶片科技實現仿生的體外三維細胞實驗模型. 2022-06-09; Available from: https://enews.nhri.edu.tw/research/7634/.
45. Carou-Senra, P., et al., Inkjet Printing of Pharmaceuticals. Adv Mater, 2024. 36(11): p. e2309164.
46. Shahzadi, L., et al., Functional Materials for DLP-SLA 3D Printing Using Thiol–Acrylate Chemistry: Resin Design and Postprint Applications. ACS Applied Polymer Materials, 2022. 4(5): p. 3896-3907.
47. Quan, H., et al., Photo-curing 3D printing technique and its challenges. Bioact Mater, 2020. 5(1): p. 110-115.
48. Mahmood, A., et al., On the Evolution of Additive Manufacturing (3D/4D Printing) Technologies: Materials, Applications, and Challenges. Polymers (Basel), 2022. 14(21).
49. Askari, A. and V. Momeni, Rheological investigation and injection optimization of Fe–2Ni–2Cu feedstock for metal injection molding process. Materials Chemistry and Physics, 2021. 271: p. 124926.
50. 財團法人國家實驗研究院儀器科技研究中心, 王. 3D金屬列印. Available from: https://wd.vghtpe.gov.tw/edupaper/files/%E5%AD%B8%E8%A8%8A%E9%9B%BB%E5%AD%90%E5%A0%B1%E7%AC%AC58%E6%9C%9F-%E7%8E%8B%E4%B8%96%E4%BB%81.
51. Tosto, C., et al., Fused Deposition Modeling Parameter Optimization for Cost-Effective Metal Part Printing. Polymers (Basel), 2022. 14(16).
52. Velásquez-García, L.F. and Y. Kornbluth, Biomedical Applications of Metal 3D Printing. Annu Rev Biomed Eng, 2021. 23: p. 307-338.
53. Group, L.U.-A.M.R. About Additive Manufacturing-Powder Bed Fusion. Available from: https://www.lboro.ac.uk/research/amrg/about/the7categoriesofadditivemanufacturing/powderbedfusion/.
54. Noor, N., et al., 3D Printing of Personalized Thick and Perfusable Cardiac Patches and Hearts. Adv Sci (Weinh), 2019. 6(11): p. 1900344.
55. Rahmani Dabbagh, S., et al., 3D bioprinted organ-on-chips. Aggregate, 2023. 4(1): p. e197.
56. Tabatabaei Rezaei, N., et al., Recent Advances in Organ-on-Chips Integrated with Bioprinting Technologies for Drug Screening. Adv Healthc Mater, 2023. 12(20): p. e2203172.
57. Liu, J., et al., Cell-laden bioink circulation-assisted inkjet-based bioprinting to mitigate cell sedimentation and aggregation. Biofabrication, 2022. 14(4).
58. Seol, Y.J., et al., Bioprinting technology and its applications. Eur J Cardiothorac Surg, 2014. 46(3): p. 342-8.
59. Ghidini, T., Regenerative medicine and 3D bioprinting for human space exploration and planet colonisation. J Thorac Dis, 2018. 10(Suppl 20): p. S2363-s2375.
60. Park, J.A., Y. Lee, and S. Jung, Inkjet-based bioprinting for tissue engineering. Organoid, 2023. 3: p. e12.
61. Shakil Arman, M., et al., Laser-induced forward transfer (LIFT) based bioprinting of the collagen I with retina photoreceptor cells. Manufacturing Letters, 2023. 35: p. 477-484.
62. Temporal Bone Anatomy and Imaging Issues, in Specialty Imaging: Temporomandibular Joint, D. Tamimi and D. Hatcher, Editors. 2016, Elsevier: Philadelphia. p. 600-605.
63. Kempfle, J.S., J.L. Turban, and A.S. Edge, Sox2 in the differentiation of cochlear progenitor cells. Sci Rep, 2016. 6: p. 23293.
64. Mizutari, K., et al., Notch inhibition induces cochlear hair cell regeneration and recovery of hearing after acoustic trauma. Neuron, 2013. 77(1): p. 58-69.
65. Scientific, T. What is the Difference Between One-Step and Two-Step RT-PCR? 2023; Available from: https://www.thermofisher.com/tw/zt/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-articles/onestep-vs-twostep-rtpcr.html.
66. McLean, W.J., et al., Clonal Expansion of Lgr5-Positive Cells from Mammalian Cochlea and High-Purity Generation of Sensory Hair Cells. Cell Rep, 2017. 18(8): p. 1917-1929.
67. Cheng, C., et al., Age-related transcriptome changes in Sox2+ supporting cells in the mouse cochlea. Stem Cell Res Ther, 2019. 10(1): p. 365.
68. Kubota, M., et al., Greater epithelial ridge cells are the principal organoid-forming progenitors of the mouse cochlea. Cell Rep, 2021. 34(3): p. 108646.
69. Hu, Y., Electroacoustic Responsive Cochlea-on-a-Chip. Adv Mater, 2024. 36(24): p. e2309002.
70. DeJonge, R.E., et al., Modulation of Wnt Signaling Enhances Inner Ear Organoid Development in 3D Culture. PLoS One, 2016. 11(9): p. e0162508.
指導教授 陳靖昀 陳信傑(Ching-Yun Chen Shin-Chien Chen) 審核日期 2024-7-29
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